Limits...
Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.

Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS, Mattson MP - BMC Dev. Biol. (2008)

Bottom Line: We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees.Glia did not appear until 4 weeks later.Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stem Cell Center, Developmental Biology, American Type Culture Collection, Manassas, VA, USA. wma@atcc.org

ABSTRACT

Background: Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.

Results: A reproducible protocol was used to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESCs. This defined adherent culture system allowed us to examine the effect of ECM molecules on neural differentiation of hESCs. hESC-derived differentiating embryoid bodies were plated on Poly-D-Lysine (PDL), PDL/fibronectin, PDL/laminin, type I collagen and Matrigel, and cultured in neural differentiation medium. We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees. Glia did not appear until 4 weeks later. Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a dose-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by the antibody against integrin alpha6 or beta1 subunit.

Conclusion: We defined laminin as a key ECM molecule to enhance neural progenitor generation, expansion and differentiation into neurons from hESCs. The cell-laminin interactions involve alpha6beta1 integrin receptors implicating a possible role of laminin/alpha6beta1 integrin signaling in directed neural differentiation of hESCs. Since laminin acts in concert with other ECM molecules in vivo, evaluating cellular responses to the composition of the ECM is essential to clarify further the role of cell-matrix interactions in neural derivation of hESCs.

Show MeSH

Related in: MedlinePlus

A homogenous population of nestin+ neural progenitors derived from a light EB. The light EB derived from TE03 cell line was plated on PDL/laminin and cultured in the NDM for 3 days. To estimate the percentage of differentiated cells expressing nestin or TuJ1, the number of labeled cells was counted from a double-immunolabeled culture at 3 days postplating for nestin (C) and TuJ1 (D), and normalized with the total number of cells determined by counting DAPI stained cells (B) overlapping phase-dark cells (A). This hESC-derived cell population consists of almost all nestin+ progenitors. TuJ1+ neurons (D, pointed by arrows) are barely seen. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570688&req=5

Figure 3: A homogenous population of nestin+ neural progenitors derived from a light EB. The light EB derived from TE03 cell line was plated on PDL/laminin and cultured in the NDM for 3 days. To estimate the percentage of differentiated cells expressing nestin or TuJ1, the number of labeled cells was counted from a double-immunolabeled culture at 3 days postplating for nestin (C) and TuJ1 (D), and normalized with the total number of cells determined by counting DAPI stained cells (B) overlapping phase-dark cells (A). This hESC-derived cell population consists of almost all nestin+ progenitors. TuJ1+ neurons (D, pointed by arrows) are barely seen. Scale bar = 100 μm.

Mentions: Human ES cell lines TE03 and TE06 were maintained and passaged weekly on mitomycin C treated mouse CF-1 embryonic fibroblasts. Colonies of hESCs were removed from feeders, triturated and re-plated in low attachment dishes to obtain spontaneously differentiating EBs. Neuroectodermal differentiation was induced in floating EBs in the neural differentiation medium (NDM). We found a marked change in appearance of differentiating EBs during culturing with the NDM. By 10 days of culture in the NDM (or 15 days of differentiation), most EBs exhibited a solid, dark core surrounded by a light band which we called "dark EBs" (Fig. 1A at 0 hr). However, if continually cultured a few more days in suspension with the NDM, some EBs gradually became transparent capsules which we called "light EBs" (Fig. 1B at 0 hr). Both dark- and light-EBs were plated onto cell culture dishes coated with PDL, PDL/fibronectin, PDL/laminin, collagen and Matrigel. Neural rosettes were readily visualized in plated EBs on all substrates except on PDL. Without clonal isolation of neural rosettes, the neuroectodermal cells in the rosettes further differentiated into neural progenitors, neurons and glia on these adherent substrates. After being plated on PDL/laminin substrates, the dark EBs generated the first nestin+ neural progenitors at 3 hours postplating, and the first TuJ1+ neurons appeared 6 hours after the nestin+ neural progenitors generated. From a dark EB, new neural precursors and neurons were constantly generated and migrated radially away from the center of aggregates, resulting in a rim of cells around the EB sphere (Figs. 1A, 2). While the dark EBs gave rise to a mixed population of nestin+ neural progenitors and TuJ1+ neurons (Figs. 1A, 2), the light EBs produced highly pure nestin+ progenitors with a few or no TuJ1+ cells (Figs. 1B, 3). In both cases, GFAP+ astrocytes and O4+ developing oligodendrocytes did not appear until 4 weeks later. Figure 1 shows time-lapse images of these two distinct differentiation patterns from dark EBs and light EBs respectively.


Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.

Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS, Mattson MP - BMC Dev. Biol. (2008)

A homogenous population of nestin+ neural progenitors derived from a light EB. The light EB derived from TE03 cell line was plated on PDL/laminin and cultured in the NDM for 3 days. To estimate the percentage of differentiated cells expressing nestin or TuJ1, the number of labeled cells was counted from a double-immunolabeled culture at 3 days postplating for nestin (C) and TuJ1 (D), and normalized with the total number of cells determined by counting DAPI stained cells (B) overlapping phase-dark cells (A). This hESC-derived cell population consists of almost all nestin+ progenitors. TuJ1+ neurons (D, pointed by arrows) are barely seen. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570688&req=5

Figure 3: A homogenous population of nestin+ neural progenitors derived from a light EB. The light EB derived from TE03 cell line was plated on PDL/laminin and cultured in the NDM for 3 days. To estimate the percentage of differentiated cells expressing nestin or TuJ1, the number of labeled cells was counted from a double-immunolabeled culture at 3 days postplating for nestin (C) and TuJ1 (D), and normalized with the total number of cells determined by counting DAPI stained cells (B) overlapping phase-dark cells (A). This hESC-derived cell population consists of almost all nestin+ progenitors. TuJ1+ neurons (D, pointed by arrows) are barely seen. Scale bar = 100 μm.
Mentions: Human ES cell lines TE03 and TE06 were maintained and passaged weekly on mitomycin C treated mouse CF-1 embryonic fibroblasts. Colonies of hESCs were removed from feeders, triturated and re-plated in low attachment dishes to obtain spontaneously differentiating EBs. Neuroectodermal differentiation was induced in floating EBs in the neural differentiation medium (NDM). We found a marked change in appearance of differentiating EBs during culturing with the NDM. By 10 days of culture in the NDM (or 15 days of differentiation), most EBs exhibited a solid, dark core surrounded by a light band which we called "dark EBs" (Fig. 1A at 0 hr). However, if continually cultured a few more days in suspension with the NDM, some EBs gradually became transparent capsules which we called "light EBs" (Fig. 1B at 0 hr). Both dark- and light-EBs were plated onto cell culture dishes coated with PDL, PDL/fibronectin, PDL/laminin, collagen and Matrigel. Neural rosettes were readily visualized in plated EBs on all substrates except on PDL. Without clonal isolation of neural rosettes, the neuroectodermal cells in the rosettes further differentiated into neural progenitors, neurons and glia on these adherent substrates. After being plated on PDL/laminin substrates, the dark EBs generated the first nestin+ neural progenitors at 3 hours postplating, and the first TuJ1+ neurons appeared 6 hours after the nestin+ neural progenitors generated. From a dark EB, new neural precursors and neurons were constantly generated and migrated radially away from the center of aggregates, resulting in a rim of cells around the EB sphere (Figs. 1A, 2). While the dark EBs gave rise to a mixed population of nestin+ neural progenitors and TuJ1+ neurons (Figs. 1A, 2), the light EBs produced highly pure nestin+ progenitors with a few or no TuJ1+ cells (Figs. 1B, 3). In both cases, GFAP+ astrocytes and O4+ developing oligodendrocytes did not appear until 4 weeks later. Figure 1 shows time-lapse images of these two distinct differentiation patterns from dark EBs and light EBs respectively.

Bottom Line: We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees.Glia did not appear until 4 weeks later.Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stem Cell Center, Developmental Biology, American Type Culture Collection, Manassas, VA, USA. wma@atcc.org

ABSTRACT

Background: Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.

Results: A reproducible protocol was used to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESCs. This defined adherent culture system allowed us to examine the effect of ECM molecules on neural differentiation of hESCs. hESC-derived differentiating embryoid bodies were plated on Poly-D-Lysine (PDL), PDL/fibronectin, PDL/laminin, type I collagen and Matrigel, and cultured in neural differentiation medium. We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees. Glia did not appear until 4 weeks later. Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a dose-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by the antibody against integrin alpha6 or beta1 subunit.

Conclusion: We defined laminin as a key ECM molecule to enhance neural progenitor generation, expansion and differentiation into neurons from hESCs. The cell-laminin interactions involve alpha6beta1 integrin receptors implicating a possible role of laminin/alpha6beta1 integrin signaling in directed neural differentiation of hESCs. Since laminin acts in concert with other ECM molecules in vivo, evaluating cellular responses to the composition of the ECM is essential to clarify further the role of cell-matrix interactions in neural derivation of hESCs.

Show MeSH
Related in: MedlinePlus